A critical barrier to progress in overcoming azole antifungal resistance in Candida albicans is the lack of a complete understanding of its molecular and genetic basis because the known mechanisms of resistance do not fully explain resistance observed among many clinical isolates. Our goal is to advance the treatment of Candida infections by identifying novel azole resistance mechanisms that can be exploited to ultimately overcome this problem. Our central hypothesis is that azole resistance in clinical isolates of C. albicans is multifactorial and involves complex genetic changes that 1) alter azole target binding, 2) activate transcriptional programs that impart resistance, and 3) reduce azole uptake. Our objectives are to 1) delineate the effects of clinically relevant mutations in ERG11, alone and in combination, on the activity of its gene product, fitness, and azole susceptibility, 2) determine the clinical significance of novel Zn(2)Cys6 transcription factors (ZCFs) that influence azole susceptibility, and 3) to discover the determinants of reduced azole import and their contribution to azole resistance in clinical isolates. Our preliminary data suggest that different ERG11 mutations diversely affect sterol demethylase activity, including alterations of catalytic efficiency, target binding kinetics, and reaction velocity. We have also observed that artificial activation of a distinct set of ZCFs in C. albicans increases azole resistance. We have identified azole-resistant clinical isolates that exhibit transcriptional profiles consistent with activation of these ZCFs and that contain candidate activating mutations in these ZCF genes. Finally, we have demonstrated that C. albicans takes up fluconazole by energy- independent facilitated diffusion. We have observed that some azole resistant isolates exhibit reduced fluconazole uptake. In Aim 1 of this proposal we will undertake genetic, microbiologic, and biochemical studies to dissect the effects of single and combinatorial mutations in ERG11 on sterol demethylase susceptibility, substrate affinity, azole binding, catalytic activity, and fitness. In Aim 2 we will undertake genetic and microbiologic studies to determine if and how mutations found in the genes encoding novel ZCFs in resistant clinical isolates result in their activation and increased azole resistance. In Aim 3 we will determine the mechanism of azole antifungal import and its contribution to azole resistance in clinical isolates of C. albicans. Our approach is innovative as we will determine for the first time precisely how mutations in ERG11 influence enzyme activity, dissect combinations of mutations, and determine the impact of such mutations on fitness of C. albicans. This work also explores novel mechanisms of azole resistance. The proposed research is significant as it will provide the understanding needed to ultimately overcome azole resistance through the development of improved azoles, interference with activated ZCFs, and enhancement of azole uptake. By fully understanding the genetic basis of azole resistance it will be possible to eventually develop non-culture based strategies to rapidly and accurately detect azole resistance in clinical isolates.